SITE-DIRECTED MUTAGENESIS OF GLUTAMATE-166 IN BETA-LACTAMASE LEADS TOA BRANCHED PATH MECHANISM

Glutamate-166 of the Bacillus licheniformis beta-lactamase was specifi cally mutated to aspartate and cysteine in order to probe the function of this residue in catalysis. In both cases, a large decrease in acti vity (k(cat)/K-m was 3.5 X 10(-5) smaller for E166C and 1 x 10(-3) sma ller for E166D than for the wild-type) was observed, although the kine tics for the two mutants were very different. The pH-rate profiles for E166D and E166C reflected the ionization characteristics of the new r esidue at site 166. This result indicates that the ionization of Glu-1 66 is responsible for the acidic limb of the k(cat)/K-m-pH profiles, a nd suggests that the function of Glu-166 is that of a general base cat alyst. The kinetics of the E166C mutant were investigated in detail. A n initial burst was observed, whose amplitude was stoichiometric with the enzyme concentration, suggesting rate-limiting deacylation of the acyl-enzyme intermediate. However, further study revealed that in the presence of 0.5 M sodium sulfate, which stabilizes the native conforma tional state, the magnitude of the burst corresponded to 2 equiv of en zyme. This observation, in conjunction with the limited effect of the mutation on K-m, indicated that the mutation resulted in a change in t he kinetic mechanism from the linear, acyl-enzyme pathway to one with a branch leading to an inactive form of the acyl-enzyme. This change i n mechanism is attributed to a substantial decrease in the rate of hyd rolysis of the normal acyl-enzyme. On the basis of a variety of observ ations, we propose that the branched pathway is characteristic of beta -lactamase catalysis when the deacylation rate is slow. The results in dicate that both acylation and deacylation are decreased by several or ders of magnitude in the E166C mutant, indicating that Glu-166 acts as a general base catalyst in both formation and hydrolysis of the acyl- enzyme intermediate.